The central theme of this proposal is the generation of force by cytoplasmic motors. Myosin, kinesin and kinesin-related proteins function in muscle contraction, cell motility and cell division. The importance of molecular motors in these key cellular functions underscores the need to understand how their force is generated and regulated. Kinesin (a molecular "shuttle" that carries cargo along microtubule "highways") will be used as a model protein in studies targeted at delineating three issues relevant to all motor proteins. . Which regions of the motor are involved in the generation of force? PCR-mediated random mutagenesis targeted at the ATP binding pocket of kinesin will be used to generate motors that have slower or faster velocities of transport relative to wild-type kinesin, a determined by the microscopy-based in vitro motility assay. . How are the two motor "heads" of kinesin and other two-headed motors- coordinated during movement along the microtubule? Heterodimeric kinesins possessing one normal and one mutant motor (generated in part !) will be constructed. The velocity and ATP hydrolysis rate of the heterodimeric kinesins will clearly distinguish between existing models for coordination. . How the activity of the motors regulated? We propose to investigate two regions of kinesin that appear to be similar to regulatory 'loop' regions in the motor myosin. We will alter the lengths of these "loops" to determine their roles in regulation.